Grid coatings for capture of proteins and other compounds

ABSTRACT

Grids comprising a coating modified with one or more capture agents and a deactivating agent are disclosed. Methods of using such grids in connection with suitable microscopy techniques, such as for determining the structure of target compounds including proteins, are also disclosed.

CROSS-REFERENCE

The present U.S. patent application is a 35 U.S.C. § 371 national phaseapplication of PCT/US16/67391, filed Dec. 16, 2016, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 62/268,737 filedon Dec. 17, 2015 and U.S. Provisional Patent Application Ser. No.62/380,586, filed Aug. 29, 2016. The contents of each of theaforementioned applications are incorporated herein by reference intheir entirety.

STATEMENT REGARDING GOVERNMENT FUNDING

This invention was made with government support under GMQ98Q17 awardedby the National Institute of Health. The government has certain rightsin the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(16063209_67374-04_UpdatedSequenceListing_27May2020ST25.txt; file size:1554 bytes; date of creation: Nov. 3, 2020) is herein incorporated byreference in its entirety.

BACKGROUND

Single particle cryo-electron microscopy analysis (SPA) is a rapidlygrowing method for elucidating structure of biological materials at nearatomic resolution due to recent advances in instrumentation andcomputational algorithms. One aspect of the SPA process that is not welloptimized, however, is sample preparation. Traditionally, proteinstargeted for structural analysis must be overexpressed and subjected totime-consuming purification and concentration steps, sometimes underharsh conditions that disrupt protein-protein interactions of interest.Recently, there have been efforts reported that seek to address theselimitations, either by improving grid rigidity to reduce beam-inducedmotion or by effecting on-grid purification with “affinity grids” thatemploy metal chelating lipids that were originally developed fortwo-dimensional protein crystallization at the lipid-water interface.The latter approach seeks to selectively capture biological targetmolecules from complex mixtures such as cell lysates as an integral partof the TEM sample preparation process.

In preparing biological samples for electron microscopy analysis,samples may be spread on an electron microscopy grid and preserved in afrozen-hydrated state by rapid freezing, often in liquid ethane nearliquid nitrogen temperature. By maintaining specimens at liquid nitrogentemperature or colder, they can be introduced into the high-vacuum ofthe electron microscope column. Most biological specimens are extremelyradiation sensitive, so they are imaged with low-dose techniques(usefully, the low temperature of cryo-electron microscopy provides anadditional protective factor against radiation damage).

Consequently, the images tend to be very noisy. For some biologicalsystems, it is possible to average images to increase thesignal-to-noise ratio and retrieve high-resolution information about thespecimen using the technique known as single particle analysis. Thisapproach in general requires that the things being averaged areidentical, although some limited conformational heterogeneity can now bestudied (e.g., ribosomes).

To better address these limitations, there have been efforts to eitherimprove grid rigidity to reduce beam-induced motion or effect on-gridpurification with ‘affinity grids” that employ metal chelating lipidsthat were originally developed for two-dimensional proteincrystallization at the lipid-water interface. The latter approach seeksto selectively capture biological target molecules from complex mixturessuch as cell lysates as an integral part of the TEM sample preparationprocess.

Although lipid monolayer affinity grids have shown some success inproducing samples for cryo-EM reconstruction at 20 Å resolution, robustperformance of the reported grid coatings is limited by film instabilityand non-uniformity under the evaporative casting methods that are oftenemployed. Additionally, these lipid films require a thin polymer layeror a holey carbon substrate layer to provide mechanical support of thedeposited film.

SUMMARY OF THE INVENTION

In one aspect of the disclosure, a grid comprising a coating modifiedwith one or more capture agents and further comprising a deactivatingagent is provided.

In a further aspect of the disclosure, a method for preparing targetsfor structure elucidation comprising contacting a grid comprising acoating modified with one or more capture agents and further comprisinga deactivating agent with a cell lysate comprising proteins andsubjecting the proteins to a suitable microscopy for structure analysisis provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A schematic illustrating sample preparation using a grapheneoxide-nitrilotriacetic acid (GO-NTA) modified TEM grid.

FIG. 2. (A) Reaction sequence for preparation of GO-NTA from GO; (B)Fourier transform infrared spectra of (i) GO, (ii) GO-NTA(O-t-Bu)₃, and(iii) GO-NTA.

FIG. 3. Pressure-area isotherm for GO-NTA sheets at the air-waterinterface, dispersed at 67 ng/mL in water at 20° C. GO-NTA sheetscompressed at a rate of 500 mm²/min.

FIG. 4. Micrographs of negatively stained his₆-T7 bacteriophage usingvarious TEM grid coatings: (A-B) GO-NTA; (C-D) PABA-GO-NTA; find (E-H)BSA-PABA-GO-NTA. Negative controls (A,C,E) demonstrate no capture ofpurified phage when Ni²⁺ is absent, whereas coatings treated with Ni²⁺(B,D,F) show capture of purified phage. Affinity capture of phage fromlysate (G) can be reversed by incubation of (G) with 500 mM imidazole(H) that removes the Ni²⁺ from the coating and abrogates the affinityinteraction between the phage and the grid surface.

FIG. 5. Micrographs of his₆-GroEL lysate affinity capture usingBSA-PABA-GO-NTA TEM grid coating. Micrographs (A-C) are negativelystained. (A) Negative control showing no capture of his₆-GroEL when Ni²⁺is absent. (B) Affinity coating activated with Ni²⁺ displays specificcapture of his₆-GroEL from lysate with (E) being an enlarged portion ofthe adjacent boxed region. Treatment of the grid in (B) with 500 mMimidazole (C) leads to Ni²⁺ stripping from the coating and abrogation ofhis₆-GroEL capture. (D) Representative cryo-EM image of affinitycaptured his₆-GroEL from lysate with (F) being an enlarged portion ofthe adjacent boxed region.

FIG. 6. (A) Class averages of his₆-GroEL images captured fromBSA-PABA-GO-NTA coated grids that were used to build the initial model;(B) Top and (C) side views of refined his₆-GroEL EM map at 8.1 Åresolution (gold standard, 0.143 criteria); (D) Fourier ShellCorrelations: conservative masking, and cross-validation curves betweenpublished GroEL map EMD-5001 and the map of the disclosure.

FIG. 7. Absorption spectra for GO-NTA as a function of concentration.

FIG. 8. Selected area electron diffraction analysis of GO-NTA film on aTEM grid.

DETAILED DESCRIPTION

While the concepts of the present disclosure are illustrated anddescribed in detail in the figures and the description herein, resultsin the figures and their description are to be considered as exemplaryand not restrictive in character; it being understood that only theillustrative embodiments are shown and described and that all changesand modifications that come within the spirit of the disclosure aredesired to be protected. Unless defined otherwise, the scientific andtechnology nomenclatures have the same meaning as commonly understood bya person in the ordinary skill in the art pertaining to this disclosure.

Novel grids with desired elasticity and rigidity to perform singleparticle analysis using electron microscopy are herein disclosed. Thegrids operate by selectively capturing targets of interest for analysiswhile not capturing contaminants or other interferants. Grids comprisingcoatings modified with at least one capture agent that can specificallytarget desired compounds, such as proteins in a biological samplethrough affinity purification, and deactivating agents to blocknon-specific binding between the grid and non-target proteins in thesame biological sample are provided. The grids herein disclosed are ableto operate at higher resolutions than previous grids without thedisadvantages in the prior art.

The grid surface components and the combination thereof enable thebenefits of the disclosure. The grids contain a coating that is modifiedwith one or more capture agents. This modification enables grids tointeract with the target of interest via the one or more capture agents.The deactivating agent or agents are present to help preventcontaminants and interferents from adhering to the grids. Such grids maythen be used in a screening assay, for example, to capture proteinscontaining a particular structure. For example, one may be interested inidentifying the structure of proteins that bind to a particularantibody. In such embodiments, the capture agents may be agrid-immobilized antibody or antibody fragments that recognizecorresponding antigens on the protein targets. Alternatively, thecapture agents could be small-molecule drug candidates that have beenattached to the grid coatings where the protein target would be capturedvia a high affinity drug-protein interaction. This would enableappropriate selection of the most promising drug candidate from acollection of compounds based on the site of engagement of thedrug-protein interaction and on the heterogeneity of the proteinscaptured, such as cell lysates.

Examples of coatings to be modified with one or more capture agentsinclude graphene, graphene oxide, micro-porous materials, nano-porousmaterials, metal-organic frameworks, activated carbon, microporouscarbon, nanoporous carbon, organic membranes, aerogels such as carbonaerogel or a metal oxide aerogel, and zeolites. The coating may be anatomic film of an element with a “low” Z (atomic number) such as carbon.The coating may be deployed in various thicknesses such as between 0.1nm and 100 μm 0.1 nm and 100 nm thick, 0.1 nm and 10 nm thick, 0.1 nmand 2 nm thick, and 0.1 nm and 1 nm thick. In such embodiments, thecoating may be a single monolayer.

In some embodiments, the coating to be modified with one or more captureagents is a carbon-based coating such as based on graphene. Specificexamples include graphene and graphene oxide (“GO”). The properties ofgraphene and graphene oxide are useful for using grids for structureanalysis. The electrical conductivity of monolayer graphene, forexample, is six orders of magnitude higher than amorphous carbon, andalthough the level of conductivity in graphene decreases with the extentof oxidation, it has been shown to recover much of this conductivityupon reduction with H₂ plasma. Additionally, unlike unsupported lipidmonolayers, the elasticity of graphene-based grids allows them to resistpermanent deformation due to mechanical transfer techniques from thematerial-water interface and such grids possess superior mechanicalstrength and conductivity. By utilizing target specificity with respectto grids of the disclosure, such as graphene-based grids, improvedstability and resistance to non-specific adsorption such that directcapture from cell lysates has been shown.

The grid can be tuned to capture various target compounds, includingproteins, based on the affinity between the target compound and thecapture agent. The modification of the coating with the one or morecapture agents may be through the formation of a covalent ornon-covalent chemical bond or by way of electrostatic forces such asthose often found between antigens and antibodies. The concept of usingcapture agents to select for compounds such as proteins is known in theart. Examples of capture agents include NTA, tris(nitrilotriaceticacid), glutathione, peptides, aptamers, antibodies, antibody fragments,or any high-affinity small molecule ligand that has an affinity to thetarget, such as a protein. Other examples of capture agents include aprotein having affinity for antibodies, Fc regions of proteins, Fabregions of proteins, or Protein A/G, nucleotides, oligonucleotides,polynucleotides, analogs of nucleotides, ATP, lectin, heparin, acarbohydrate, ubiquitin, SNAP tag, glucosaminoglycan, chitin, oramylase. Table 1 represents examples of several known affinityinteractions between capture agents and their various associated targetson proteins. Any of these capture agents may be used in connection withthe associated targets with grids of the disclosure for structureanalysis or elucidation, for example.

TABLE 1 Affinities Between Capture Agents and Targets Capture AgentAssociated Target Nickel(II) or Cobalt(II):NitrilotriaceticPolyhistidine fusion acid Glutathione Glutathione S-transferase fusionAlkyne Azide-containing target Azide Alkyne-containing target MaleimideThiol or amine-containing target Biotin Avidin, Streptavidin,NeutrAvidin ® (a deglycosylated version of avidin), CaptAvidin ™ (aprotein including a nitrated tyrosine in its biotin-binding site)Avidin, Streptavidin, NeutrAvidin ® Biotin fusion or modification (adeglycosylated version of avidin), CaptAvidin ™ (a protein including anitrated tyrosine in its biotin-binding site) Antibody Antigen Antibodyfragment Antigen Protein A, G or L Antibody anti-HA antibody or fragmentHA (YPYDVPDYA (SEQ ID NO: #1)) fusion anti-Myc antibody or fragmentc-Myc (EQKLISEED (SEQ ID NO: #2)) fusion ANTI-FLAG ® (an antibody toFLAG ® (a polypeptide tag FLAG ®) antibody or fragment having thesequence motif DYKDDDDK) (SEQ ID NO: #3)) fusion anti-V5 antibody orfragment V5 (GKPIPNPLLGLDST (SEQ ID NO: #4)) fusion Maltose Maltosebinding protein (MBP) fusion Halo HaloTag fusion ® (a polypeptide tagderived from a bacterial haloalkane dehalogenase wherein the tag iscomposed of two covalently bound segments including a haloalkanedehalogenase and a synthetic ligand of choice: Promega Corporation)fusion Dibenzocyclooctyne Azide-containing target N-HydroxysuccinimideThiol or amine-containing target Amine Amine-reactive targetNi²⁺:NTA-poly(ethylene glycol Polyhistidine fusion2000)-1,2-distearoyl-sn-3- glycerophosphoethanolamine

Common capture agents that are covalently bound to the coating arechelating agents such as poly carboxylic acids. Specific capture agentsinclude ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), diaminocyclohexanetetraaceticacid (DCTA), nitrilotris(methylene)triphosphonic acid (NTTA),2-aminoethanethiol, thiobis(ethylenenitrilo)tetraacetic acid (TEDTA),N-2-acetamidoimino-diacetic acid (ADA), iminodiacetic acid (IDA),hydroxamic acid, carboxymethylated aspartate ((VIA) and nitriiotriaceticacid (NTA), tris(nitrilotriacetic acid), N¹,N⁴,N⁸-tris(nitriiotriaceticacid)-1,4,8,11-tetraazocyclotetradecane and analogs or homologs thereof.When performing as capture agents in grids of the disclosure, thechelating agents are typically chelated to a metal ion. Examples of suchions include Cu²⁺, Ni²⁺, Co²⁺, Ca²⁺, Mg²⁺, Cd²⁺, Gd²⁺, Ru²⁺, and Fe²⁺.

Table 2 illustrates certain capture agent-coating modifications, thenature of the interaction with the target protein of interest, theaffinity tag used for the protein, the pH at which the chemistryoperates and some relevant application.

TABLE 2 Non-covalent Capture Method Capture Agent Specificity Working pHApplications IMAC NTA, Ni2+-activated Histidine tag 2-14 Recombinant(can be activated Histidine-tagged with other metal ions) proteins GSTGlutathione GST tag 1-14 Recombinant GST-tagged Proteins Protein AProtein A IgG 2-10 IgG-mediated capture of target protein BiotinStreptavidin Biotin 4-10 Biotinylated protein

Table 3 is similar to Table 2 except it illustrates certain covalentcapture agent-coating interactions.

TABLE 3 Covalent Capture Method Capture Agent Specificity Working pHApplications NHS Amidation Lysine 3-11 Capture at surface-exposed lysineresidues Maleimide Thioether Cysteine 4-10 Capture at surface-exposedcysteine residues Cyclooctyne “Click” reaction Azide 4-10 Azidatedprotein Boronate Boronate ester cis-diols 2-10 Capture of any targetmolecule containing a cis-1,2-diol Amine Amidation (after Aspartate,2-10 Capture at EDCI activation) Glutamate surface-exposed carboxylates

Grids of the disclosure further comprise deactivating agents. Suchdeactivating agents defoul the surface of the grid to optimize analysisof the target by minimizing interference from other compounds andproteins. Without being bound by theory, it is believed that thedeactivating agents tightly pack on the grid to remove binding locationsfor compounds or other proteins that are not captured by the captureagents. Thus, deactivating agents aid in the selectivity of the overallaffinity of the grid and improve signal to noise by blockingnon-specific target capture by the grid. Example of deactivating agentsinclude, but are not limited to, water soluble polymers such aspolyethylene glycols. Other examples include dextran, agarose,methoxy-poly(ethylene glycol350)-1,2-distearoyl-sn-3-glycerophosphoethanolamine, albumin, bovineserum albumin, casein, or other protein, small molecule, or hydrophilicpolymer that blocks non-specific absorption such as from proteins orother compounds.

In some embodiments, more than one deactivating agent may be used. Forexample, the deactivating agent 4-aminobenzoic acid (PABA) may bechemically bound to the chemically modified coating. In thoseembodiments, a second deactivating agent, such as BSA, may also be usedwhich is not so chemically bound.

The targets of the disclosure may be one or more proteins or othercompounds. For example, one may use grids to capture organic moleculesin a screen. A typical use, however, is to capture one or more targetproteins of interest for structure analysis by electron microscopy. Theproteins may or may not be tagged for such purposes. The proteins may beprepared in any number of conventional ways such as from a celllysate—which may be clarified prior to capture with the grids of thedisclosure. Cell lysates are often E. coli, plant, or human celllysates. The capture agent is chosen so that it is selective for theinteraction of interest between the capture agent and the targetprotein. It is known in the art to tag proteins with polyhistidine andcompounds that are selective for polyhistidine include NTA. For example,polyhistidine tags (or other peptide tags such as FLAG® (a polypeptideprotein tag having the sequence motif DYKDDDDK) or protein fusions suchas Glutathione S-transferase) are typically engineered into anexpression plasmid for the protein and transfected into E. coli suchthat the N- or C-terminus of the expressed protein bears the affinitytag. Thus, in some embodiments, the proteins of interest are taggedusing known techniques in the art with polyhistidine wherein the gridcomprises GO-NTA or GO-NTA-PABA as a coating. Other tags includeGlutathione S-transferase, Avidin, Streptavidin, NeutrAvidin® (adeglycosylated version of avidin), CaptAvidin™ (a protein including anitrated tyrosine in its biotin-binding site) biotin, an antigen, anantibody, HA (YPYDVPDYA (SEQ ID NO: #1)), c-Myc (EQKLISEED (SEQ ID NO:#2)), FLAG® (a polypeptide protein tag having the sequence motifDYKDDDDK) (SEQ ID NO: #3)), V5 (GKPIPNPLLGLDST (SEQ ID NO: #4)), Maltosebinding protein, artificial amino acid modifications (e.g., alkyne orazide) or HaloTag® (a polypeptide tag derived from a bacterialhaloalkane dehalogenase wherein the tag is composed of two covalentlybound segments including a haloalkane dehalogenase and a syntheticligand of choice; Promega Corporation). When so prepared, the proteintarget, immobilized on the surface as an ensemble of single particleswith different orientations with respect to the surface normal, may beimaged or otherwise analyzed with a suitable microscope such as anelectron microscope or a Raman microscope. When the grid coatingmodified with the one or more capture agents is sufficiently thin, suchas with a monolayer, the target may be presented in the same focal planeof the microscope, thus providing the advantage that both target andmodified coating be in focus during image capture which is advantageousfor obtaining high resolution structures.

Examples of resolutions obtained by the grids of the disclosure includeresolutions to between 1 Å and 4 Å including between 1 Å and 4 Å andbetween 3 Å and 4 Å. In other embodiments, the resolution is between 1 Åand 10 Å including between 4 Å and 10 Å, 5 Å and 10 Å, 6 Å and 10 Å, 7 Åand 10 Å, 8 Å and 10 Å, and 8 Å and 9 Å and all values in between forthese examples.

Structural analysis of proteins may be performed by contacting the gridsof the disclosure with cell lysates comprising those proteins and thensubjecting the proteins to a suitable microscopy, such as electronmicroscopy, including cryo-electron microscopy, transmission electronmicroscopy, or scanning electron microscopy. In some embodiments, theproteins are tagged with an affinity tag with example of such a tagbeing a polyhistidine tag. In some of these embodiments, a coating ofgraphene or GO may be modified with a chelating agent and a metal ionsuch as NTA and Ni²⁺ and a PABA deactivating agent. The resolutionobtained in these embodiments may be between 1 Å and 4 Å includingbetween 2 Å and 4 Å and between 3 Å and 4 Å. In other embodiments, theresolution is between 1 Å and 10 Å including between 4 Å and 10 Å, 5 Åand 10 Å, 6 Å and 10 Å, 7 Å and 10 Å, 8 Å and 10 Å, and 8 Å and 9 Å andall values in between for these embodiments. When the grid is used in anelectron microscope for analysis, it may be placed in a sample holdersuch as made out of a nonferrous metal.

In various embodiments of the disclosure, the coating comprises one ormore graphene or GO sheets modified with one or more capture agents.When graphene or GO is modified with one or more capture agents, acommonly used capture agent, especially with polyhistidinetagged-proteins is Nα, Nα-dicarboxymethyllysine (which is the same asnitrilotriacetic acid and referred to herein as NTA).

In various embodiments, the coating may be modified with one or morelipid monolayers supported by a thin carbon or a graphene-type layer orby single wall carbon nanotubes. In these embodiments, depending on thestructure of the monolayer, it may serve both as a capture agent and asa blocking agent. For example, a Ni-chelated NTA bound monolayer may actas a capture agent, even if bound to a polyethylene glycol group as inthe case of Ni²⁺:NTA-poly(ethylene glycol2000)-1,2-distearoyl-sn-3-glycerophosphoethanolamine in Example 21. Inthat Example, methoxy-poly(ethylene glycol350)-1,2-distearoyl-sn-3-glycerophosphoethanolamine acts as adeactivating agent.

In other embodiments, single wall carbon nanotubes are coated with amonolayer coating comprising a capture agent being one or more ofNTA-PEG2000-DSPE or NTA-PEG-2000-DTPE and a deactivating agent such asmPEG350-DTPE.

In some embodiments, GO-modified grids are prepared, which have lowerbackground signal and improved conductivity (which aids in combattingsample charging and instability during image capture) than previouslipid monolayer coated affinity grids. Such grids may be modified, forexample, by covalently linking the grid coating to a chelating agent,such as N^(α), N^(α)-dicarboxymethyllysine to make GO-NTA. The GO-NTAmay then be chelated to a metal ion such as Cu²⁺, Ni²⁺, Co²⁺, Ca²⁺,Mg²⁺, Cd²⁺, Gd²⁺, Ru²⁺, or Fe²⁺. A common metal ion used is Ni²⁺.

In one example, a GO-modified grid is shown in FIG. 1. In process step(i), a GO-NTA monolayer is deposed onto a transmission electronmicroscopy (TEM) grid via Langmuir-Schaefer (L-S) transfer. In step(ii), activation of NTA occurs with Ni²⁺ (referred to as Ni(II) inFIG. 1) (or another divalent metal ion can be used). The blocking ofnon-specific reaction and/or adsorption sites with 4-aminobenzoic acid(PABA) and bovine serum albumin (BSA) occurs in step (iii). Incubationof clarified lysate containing the target with the blocked grid in step(iv), and the washing of non-target molecules (impurities) from thegrid, followed by cryo-fixation or staining occurs in step (v). Usingthe general procedures illustrated in FIG. 1, both His₆-T7 bacteriophageand His₆-GroEL were selectively captured from cell lysates by anickel-chelated single monolayer GO-NTA grid using bovine serum albumin(BSA) and PABA as deactivating agents. In the case of His₆-GroEL,multiple single particle analyses were performed on the protein with thehighest resolution being at 8.1 Å.

EXAMPLES Example 1

General Description of Functionalization of GO Sheets with NTA. GO wasproduced from grapheme using Hummer's method. Activation of the GOcarboxylic acid groups with SOCl₂ prior to reaction with thetris-t-butyl ester of lysine NTA gave GO-NTA-(O-t-Bu)₃. TFA deprotectionof this intermediate gave GO-NTA (FIG. 2A). Fourier transform infraredspectroscopy was used to monitor these reactions as shown in FIG. 2B.The spectra of GO displayed a broad absorption at 3236 cm⁻¹ (O—Hstretch) and a sharper absorption at 1648 cm⁻¹ (C═O stretch). The NTA-GOtris-t-butyl ester displayed an additional absorption at 2933 cm⁻¹ (C—Hstretch) due to the incorporation of the lysine and t-butyl moieties.Following treatment of NTA-GO tris-t-butyl ester with TFA, the presenceof the aliphatic C—H stretching was greatly reduced, indicatingsuccessful deprotection of the NTA chelator substituents.

Previous work has shown that the typical GO sheet absorption band at˜240 nm is shifted to ˜270 nm when the GO sheets are dispersed inaqueous solution. The origin of this hypsochromic shift is due to n-π*electronic transitions arising from the C═O bonds introduced byoxidation. GO-NTA samples prepared in this manner exhibited a majorabsorption peak at ˜280 nm (FIG. 7), in good agreement with thesereports.

Example 2

Graphene-Oxide Synthesis. GO was synthesized using an improvedHummers'method that is easier to execute, is higher yielding, and doesnot evolve toxic gases. It has been reported that there is no decreasein conductivity in the final product between the original and improvedmethod, making it an attractive route for large scale production of GO[Marcano, D. C., et al. ACS Nano 2010 4, 4806-4814], When a 9:1 mixtureof H₂SO₄ and H₃PO₄ (130 mL total volume) was stirred with 1 g ofgraphite flakes (F516 flake graphite, 200-300 mesh, Asbury Carbons,Inc.) and KMnO₄ (6.0 g, 6.0 wt. equiv.), the reaction began with heatingto ˜40° C. and proceeded with further heating and stirring at 50° C. for12 hours before cooling to 20° C. and pouring the reaction mixture into120 mL of ice cold-water with 1 mL 30% H₂O₂. Next, this suspension waspassed through a metal U.S. Standard testing sieve (W. S. Tyler, 300 μm)and then passed through a glass wool plug to filter larger particulates.The filtrate was then centrifuged at 4,000 rpm for 4 h, the supernatantdiscarded, and the pellet washed twice with a 1:1:1 volumetric ratio ofH₂O, 30% HCl, and EtOH before passing the material through the testingsieve and centrifuging the filtrate at 4,000 rpm for 4 h to pellet theaggregated material. The supernatant was precipitated with Et₂O (200 mL)and filtered through a 0.45 μm PTFE membrane to gather the solid. Thefinal material was dried under a 15 μm vacuum for 12 h, yielding 1.8 gof GO.

Example 3

Graphene-Oxide-NTA Synthesis, GO was synthesized as described by Marcanoet al. This intermediate (335 mg) was stirred in a mixture of SOCl₂ (60mL) and DMF (1.5 mL) at 70° C. for 3 d before evaporating the SOCl₂ andDMF and washing the residue with dry DCM (3×50 mL). ACN (50 mL) and Et₃N(3 mL) were then added and the mixture stirred for 30 min.Tris(O-t-butyl)-N^(α),N^(α)-dicarboxymethyllysine ester (533 mg) wasthen added and the mixture stirred at 100° C. for 3 d before washingwith THF and H₂O (9,000 rpm for 15 min, 3 times for each solvent),before vacuum drying at 60° C. for 24 h, TFA (10 mL) in THF (30 mL) wasadded to the dried t-butyl-NTA ester intermediate (180 mg) and stirredat 60° C. for 5 h before washing with THF and H₂O (11,000 rpm×15 min, 3times for each solvent).

Example 4

GO-NTA Exfoliation. The GO-NTA sheets from Example 3 were ultrasonicallyexfoliated at 1 mg/mL by suspension of the powder in 5:1 MeOH:H₂O usingprobe sonication at 150 watts for five cycles (45 s sonication followedby 45 s of rest in each cycle). The product was centrifuged at 1200 gfor 10 min, after which the supernatant of exfoliated GO-NTA sheets wasremoved from the sediment of aggregated sheets and subjected to another5 rounds of sonication. A final centrifugation at 1200 g for 10 min wasperformed prior to removal of the supernatant to yield a GO-NTA solutionthat was stored for subsequent grid coating experiments.

Example 5

Langmuir-Trough Setup. Exfoliated GO-NTA was deposited at the air-waterinterface of a Kibron μTrough via a syringe pump fitted with a 20 mLsyringe. The GO-NTA dispersion was loaded into the syringe and slowlyintroduced at the air-water interface at a rate of 100 μL/min until thesurface pressure reached 15 mN/m. The film was then allowed to relax for5 min, followed by slow compression of the film to 15 mN/m. IPA was thenadded to the subphase and the film transferred to either Si wafers, bare1500 mesh TEM grids, or holey carbon grids by L-S transfer.

Example 6

GO-NTA Monolayer Formation. Compression of the GO-NTA material at theinterface gave a characteristic surface pressure-area isotherm (FIG. 3),suggesting a progression from isolated GO-NTA sheets to closeedge-to-edge packing of GO-NTA sheets, followed by folding, wrinkling,and sliding of the nearest neighbor GO-NTA sheets atop one another uponfurther compression, in a manner analogous to pressure-induced collapseof Langmuir phospholipid monolayer films. Repulsive electrostaticinteractions and attractive van der Waals forces compete as GO-NTAsheets come into close contact. Previous work with GO monolayers hassuggested that over-compression of GO causes irreversible coagulationabove ˜15 mN/m due to the increasing participation of attractive van derWaals interactions once the repulsive electrostatic interactions betweensheet edges has been overcome by lateral compression. Transfer of thesefilms onto silicon substrates at multiple surface pressures enabled thetransfer of single layer GO sheets at surface pressures above 15 mN/m.

Example 7

4-Aminobenzoic acid (PABA) Modification of GO-NTA. GO-NTA (1 mg/mL) waspartially deactivated by adding PABA (30 mg) to a 10 mL GO-NTAdispersion. This mixture was probe sonicated at 150 W for 30 sec ofcontinuous sonication, followed by shaking for 24 h on a rotary mixer.The PABA-GO-NTA was then exfoliated and washed as described above forGO-NTA exfoliation.

Example 8

Fluoresced Modification. Fluorescein modification of GO-NTA wasperformed by adding 2 mg of aminofluorescein to an aqueous solution ofPABA-GO-NTA (10 mL at 1 mg/mL). This mixture was probe sonicated for 30s at 150 W of continuous sonication and then placed on a rotary mixer inthe dark for another 24 h. The material was then centrifuged to pelletthe GO species before re-suspending in water, addition of 5:1 MeOH:H₂O,re-pelleted, and decanted a total of 10 times before exfoliation of theFluorescein-PABA-GO-NTA (F-PABA-GO-NTA) product as described above forGO-NTA.

Example 9

Bovine Serum Albumin (BSA) Modification. Following L-S transfer ofGO-NTA or PABA-GO-NTA onto EM grids and overnight drying in adesiccator, the grids were placed on a strip of Teflon before additionof BSA (10 μL of 0.1 mg/mL) and incubation for 5 min, followed by 3×20μL double deionized H₂O washes. The modified grids were then stored in adesiccator until use.

Example 10

Fluorescence Microscopy Sample Preparation. F-PABA-GO-NTA was depositedonto 1500 mesh grids in the dark by L-S transfer as described above.After transfer, the grids were allowed to dry in the dark for 1 d beforesandwiching them between a glass and cover slip with 5 μL of doubledeionized H₂O and the sandwich sealed with nail polish. The glass slidewas then mounted on a light microscope for epifluorescence imaging.

Example 11

GO Concentration Measurements. The concentrations of the GO-NTAdispersions were measured at different steps of the synthesis bymonitoring the UV-vis spectra of the products. Since each batch ofGO-NTA has minor differences in concentration, each preparation wasevaluated for its own experimentally determined extinction coefficientfor subsequent concentration measurements. Standard solutions used todetermine the extinction coefficients were prepared by dispersing aweighed amount of dry GO-NTA into known volumes of 5:1 MeOH:H₂O andmeasuring the absorbance at 280 nm across a series of dilutions with 5:1MeOH:H₂O. The extinction coefficient was derived from the slope of theseconcentration-dependent absorption plots.

Example 12

GO-NTA Grid Treatment with Purified His₆-T7 Bacteriophage. PurifiedC-terminal gp10 His₆-T7 bacteriophage was initially prepared at aconcentration of 10¹² particles/mL, with dilution to 10¹⁰ particles/mLin HEPES buffer (pH=7.4) before application to the affinity gridsurface. GO-NTA modified grids were placed on a Teflon strip, 1 mM NiSO₄(10 μL) added and the grids incubated for 5 min before washing withdouble deionized H₂O (2×20 μL) and HEPES buffer (1×20 μL). Purifiedphage (3.5 μL) was then applied to the surface and incubated for 2 minbefore washing with HEPES (2×20 μL), double deionized H₂O (1×20 μL), andstaining with 2% uranyl acetate (5 μL).

Example 13

GO-NTA Grid Treatment with His₆-T7 Bacteriophage Lysate. BL21 bacterialcells in 1 mL of LB media were grown to an OD of 0.8 before adding 1.0μL of His₆-T7 bacteriophage (1×10¹² particles/mL) to the media andshaking the culture for 1 h. After bench top centrifugation of thecells, the supernatant was withdrawn for use in His₆-T7 bacteriophageparticle capture studies. The grids were Ni²⁺-activated as describedabove, except that His₆-T7 lysate (5 μL) was applied to the surfacebefore incubation for 2 min. The grids were then washed with HEPES (2×20μL), double deionized H₂O (1×20 μL), and then stained with 2% uranylacetate (5 μL).

Example 14

GO-NTA Grid Treatment with His₆-GroEL Lysate. The ASKA Library was usedto express N-terminal His₆-GroEL. Cells containing N-His₆-GroEL geneoverexpression vector were grown to OD=0.6 (in 100 mL of LB broth usinga 37° C. shaker/incubator) and induced with a final concentration of 1.0mM IPTG, before allowing the cells to grow for an additional 4 h. Aftercentrifugation and removal of the supernatant, the cell pellet wasre-suspended in lysis buffer (20 mM HEPES, 100 mM NaCl, pH=7.4, 100 μgaprotinin, 174 μg phenylmethanesulfonyl fluoride (PMSF), and 500 μg oflysozyme) and allowed to sit for 20 min. Further disruption of the cellmembranes was effected by 110 W probe sonication (35 pulses at 1second/pulse), followed by centrifugation at 11,000 g for 10 min. Thesupernatant containing His₆-GroEL was diluted 10-fold and assayed foraffinity-binding using the Ni²⁺-activation procedure described above,except that N-His₆-GroEL lysate (5 μL) was applied to the surface andincubated for 2 min. The grids were then washed and stained with 2%uranyl acetate as described above.

Example 15

Affinity Capture of His₆-GroEL from E. coli Lysates onto BSA-PABA-GO-NTAGrids for Cryo-EM Imaging. Samples were prepared as described above fornegative stain TEM imaging, except that BSA-PABA-GO-NTA modified gridswere exposed to His₆-GroEL lysate, after which the excess solution wasremoved by blotting twice for 6 s per blot using an offset setting of −1at 80% humidity using a Vitrobot device (FEI Company). The grids werethen plunged into liquid ethane for cryofixation and imaged at 300 kV onan FEI Titan Krios equipped with a Gatan K2 Summit direct electrondetection camera using low-dose techniques. Integrated microscopeautomation software Leginon was used to acquire a large set ofmicrographs at 11,000× magnification with an exposure time of 7.6 sec.

Example 16

Single Particle Analysis of His₆-GroEL. Direct electron detector movieframes were processed in Appion to produce a set of averaged,motion-compensated micrographs to be used in subsequent steps. Themicrographs had a 1.32 Å²/pixel resolution across a 4096 Å×4096 Å array.EMAN 2.1 software was used for reconstruction of 5363 particles thatwere manually picked from 217 micrographs using a box size of 256.Automatic contrast transfer function (CTF) estimation and structurefactor were determined from the incoherent sum of particles using e2ctfand phase-flipped to generate high-pass CTF-corrected particle stacks.Defocus was estimated to range between 0.4 μm-4 μm, but 55% of theparticles were defocused between 2-3 which resulted in a somewhat jaggedCTF slope. Particles were binned 2× for class averaging and 12 classeswere chosen to create an initial model with imposed D7 symmetry. Theclasses contained a mix of top and side views. In the refinement steps,the input set of particles was divided into even/odd halves, eachcontaining 2682 particles. Two independent refinements were generated,producing a gold standard of 8.1 Å (using 0.143 criteria) after 12iterations over two refinements with an angular sampling of 1.76degrees. Additionally, a Fourier shell correlation against an existinghigh-resolution cryo-EM map, EMD-5001 was performed. The maps wererotated and translated using Chimera to fit the volumes together. Thecon-elation of this model against EMD-5001 (4.2 Å) gave an approximateresolution of 9 Å.

Example 17

Characterization of GO-NTA Monolayers. Epifluorescence microscopy, AFM,and SEM was employed to determine the thickness and lateral distributionof GO-NTA sheets deposited onto solid substrates by L-S transfer fromthe air-water interface. In particular, epifluorescence microscopy ofF-PABA-GO-NTA monolayers revealed a monolayer-coated grid and negativecontrol bare Cu TEM grid samples that showed significantly greaterfluorescence intensity for grids coated with F-PABA-GO-NTA.

SEM and AFM analyses were performed after compression to 15 mN/m and L-Stransfer of GO-NTA monolayer sheets onto Si wafers. To prepare Si wafersfor L-S transfer, ˜2.25 cm² wafers were cut and glued (bottom side) ontoa transfer tube. The surface pressure was maintained until the Si wafercontacted the monolayer; the film was then recompressed to 15 mN/m afterthe L-S transfer step. The area difference before and after L-S transferindicated transfer efficiencies of 75-85%. Image analysis of the coatedSi wafers revealed the presence of GO-NTA monolayer sheets transferredfrom IPA-containing subphases with ˜1.3 nm thicknesses that wererelatively uniform, in good agreement with previously reported valuesfor single layer GO. In the absence of IPA; however, data from SEM andAFM experiments revealed GO-NTA films comprised of overlapping sheetsand undesirable layer thickness variations.

Selected area electron diffraction analysis of GO-NTA L-S films,deposited onto bare 2000 mesh grids from the air-IP A-THbO interface,revealed a hexagonal diffraction pattern, indicative of a single layerof graphenic material. The measured intensity of the inner (2,3) andouter (1,4) peaks confirms the presence of a single GO-NTA layer (FIG.8).

Example 18

Affinity Capture of His₆-T7 Bacteriophage from E. coli Lysate UsingGO-NTA Monolayer Purification and PABA+BSA as Antifouling Agents. Thecapacity of GO-NTA coated grids to capture His₆-T7 bacteriophage(His₆-T7) by affinity interaction was examined first by negative-stainTEM. After a 2 min exposure of purified His₆-T7 on GO-NTA modified 1500mesh grids, dense clusters of phage particles were found on the GO-NTAsurface in the absence of Ni²⁺ (FIG. 4A). GO-NTA was then treated with4-aminobenzoic acid (PABA) after L-S transfer. The resulting PABA-GO-NTAgrids showed a reduction in, but incomplete abrogation of, non-specificHis₆-T7 binding under the same incubation conditions (FIG. 4C), Whenactivated with Ni²⁺, PABA-GO-NTA grids revealed a higher density ofphage particles due to engagement of the NTA:Ni²⁺:His₆ affinityinteraction (FIG. 4D). The PABA-GO-NTA grids were incubated with BSAimmediately before the affinity capture step. Under these conditions,BSA appears to complete the blockade of non-specific viral particleadsorption (FIG. 4E), suggesting that BSA inhibits non-specific bindingmore effectively here than PABA modification alone. After Ni²⁺activation of the BSA-blocked PABA-GO-NTA surfaces, a recovery inHis₆-T7 binding to the grids (FIG. 4F) was observed. To furtherdemonstrate the Ni²⁺ dependence of this interaction, the grid wastreated with 500 mM imidazole, which removes the Ni²⁺ ion, leading tothe elution of His₆-T7 from, the grid. Taken together, these findingsdemonstrate the importance of deactivating highly reactive chemicalfunctionalities on the surface of GO prior to use in affinity captureexperiments.

Next, it was attempted to capture His₆-T7 directly, in that noadditional processing other than centrifugation was used to removeundissolved components, from clarified E. coli lysate. The engineeredHis-tag does not interfere with His₆-T7 infectivity, thereby enablingthe infection of BL21 cells and viral replication in vitro. A negativecontrol experiment demonstrated that Ni²⁺-free BSA-PABA-GO-NTA gridsresulted in little or no capture of bacteriophage and minimal backgroundadsorption from non-targeted cellular material; however, Ni²⁺ activationprompted selective His₆-mediated binding of bacteriophage to the gridsurface (FIG. 4G). As an additional control, the grid was washed with500 mM imidazole after Ni²⁺, but prior to incubation with lysate, todemonstrate that imidazole stripping of the metal ion results in theabrogation of His₆-T7 binding (FIG. 4H), These results indicate thatBSA-PABA-GO-NTA coated grids are able to capture His₆-T7 directly fromclarified lysate on the grid using the NTA:Ni²⁺:His₆ affinityinteraction.

Example 19

Affinity Capture of GroEL From E. coli Lysate Using BSA-PABA-GO-NTAMonolayer Purification. The performance of antifouling BSA-PABA-GO-NTAcoatings for high-resolution single particle reconstruction analysis wasevaluated by performing on-grid affinity capture of His₆-GroEL from E.coli lysates. As observed for His₆-T7 capture, specific binding ofHis₆-GroEL occurred only with Ni²⁺-activated (FIG. 5B), but notNi²⁺-free (FIG. 5A) or 500 mM imidazole treated grids (FIG. 5C). Cryo-EMimages of His₆-GroEL deposited onto BSA-PABA-GO-NTA coated grids wereobtained (FIG. 5D). Initial attempts at His₆-GroEL capture andcryofixation on 1500 mesh grids coated with BSA-PABA-GO-NTA generatedunacceptably thick sample vitrification; however, high quality samplesof His₆-GroEL captured from lysate were afforded by BSA-PABA-GO-NTAfilms deposited by L-S transfer onto lacey carbon-supported 400 meshcopper grids.

Example 20

Single Particle Analysis of His₆-GroEL. EMAN 2.1 was used for singleparticle analysis of His₆-GroEL deposited onto BSA-PABA-GO-NTA coatedgrids since this protein target is often used for gauging workflowperformance and data processing robustness. The reconstruction effortfollowed the usual steps from within the application, except that theparticles were manually picked. Background signal contributions by theBSA blocking layer may also have contributed to the difficultiesencountered during attempts at automated particle picking. Nonetheless,5363 particles were hand-picked from 217 micrographs and the particlesrapidly converged into coherent classes displaying high contrast (FIG.6A).

Of 50 total class averages, 12 were chosen to produce an initial modelwith imposed D7 symmetry (FIG. 6A). After 12 refinement iterations withan angular sampling of 1.76 degrees, we were able to produce a goldstandard (0.143 criteria) density map having 8.1 Å resolution usingconservative masking (FIGS. 6B-D). There were visible nodes in the FSCcurve at regular intervals that resulted from an uneven distribution ofmicrograph defocuses.

To verify the accuracy of the model, a comparison with published 4.2 Åresolution cryo-EM map EMD-5001 that was also produced by EMAN using D7symmetry. Chimera was used to fit the volumes before calculating theFSC, yielding a 9 Å resolution using 0.5 criteria (FIG. 6D). As noted inFIG. 6D, the conservative masking and the cross validation curves aregood fits at 0.5 FSC and 0.143 FSC, which are accepted standards in thefield.

A substantial difference between the map and the published structure wasobserved, wherein additional electron density within the inner pore ofthe protein was found in the case of the His₆-GroEL map. This finding isattributable to the extended ammo acid sequences at theN- and C-termini(i.e., MRGSHHHHHHTDPALRA (SEQ ID NO: #5) and GLCGR (SEQ ID NO: #6),respectively) of our His₆-GroEL construct derived from the ASKA Library,which is not present in the published structure. When taking thatdifference into account, however, the fit was good as indicated in FIG.6D.

Wild type N- and C-termini of the protomers are located at the surfaceof the inner pore lining the assembled tetradecameric complex. Thus, the14 engineered subunits comprising the His₆-GroEL complex yield anadditional 308 residues that occupy the pore, of which 84 arehistidines. Given the large number of potential metal chelationregioisomers and topoisomers, as well as the high potential forconformational flexibility in the N- and C-terminal sequences, thisdensity is unlikely to adopt a defined structure and instead appears asa filled “droplet” within each ring. Also, there is a noticeabledecrease in density in the flexible apical region that suggests lessstructural coherency. From these findings, it is inferred that theadditional pore residues, along with NTA chelation, may create dynamicdistortions to the structure of GroEL that could vary for each particle,reducing coherency and map density at the apical ends.

Example 21

Langmuir-Schaefer Film Deposition Onto Graphene-bearing Grids: Stocksolutions of two lipid mixtures (e.g., Ni²⁺:NTA-poly(ethylene glycol2000)-1,2-disteaxoyl-sn-3-glycerophosphoethanolamine (capture agent) andmethoxy-poly(ethylene glycol350)-1,2-distearoyl-sn-3-glycerophosphoethanolamine (deactivatingagent)) in proportions appropriate for the desired surface density oftarget protein (e.g., 1:99-1:5 capture ligand lipid:blocking lipidratio) are prepared in CHCl₃ at 2.0 mg/mL and stored at −80° C. Thesesolutions are spread at the air-water interface of a monolayer troughvia 10 μL microsyringe and compressed to a final surface pressure of30-45 mN/m. The compressed lipid monolayers are then deposited ontoGraphenea Quantifoil Gold TEM grids or other suitable TEM grid bearing agraphene monolayer by Langmuir-Schaefer transfer. The LS film was thendried and transferred to a standard TEM grid box for later use.

Example 22

General Guidelines for Sample Incubation on Affinity Grids. The observedsample capture efficiency is a function of the target proteinconcentration, the target protein molecular weight, and the surfacedensity of capture ligand. Approximate incubation times at 25° C. for agiven target protein concentration are summarized below; however, theactual times required for optimal binding will depend on total proteinconcentration, sample viscosity, competing ligand concentrations, andsample incubation temperature.

0.1 mg/mL 0.5 mg/mL 1.0 mg/mL 2 mg/mL 35 kD Target 1% capture agent 15min 10 min 5 min 2 min 5% capture agent  5 min  2 min 1 min 30 sec   100kD Target 1% capture agent 20 min 14 min 7 min 4 min 5% capture agent  7min  4 min 2 min 1 min 300 kD Target 1% capture agent 30 min 20 min 10min  5 min 5% capture agent 10 min  7 min 5 min 3 min ≥1 mD Target 1%capture agent 45 min 35 min 25 min  15 min  5% capture agent 15 min 12min 8 min 5 min

Example 23

Synthesis of Specific Compounds—General. Materials. Solvents werepurchased from Mallinckrodt/Baker and used without further purificationunless noted. Toluene was purchased from Fisher.N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) andN^(ε)-((benzyloxy)carbonyl)-1-lysine (H-Lys(Z)—OH) were purchased fromAdvanced Chemtech. Heterobifunctional PEG derivatives were purchasedfrom JenKem technology USA.1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and1,2-(tricosa-10′,12′,-diynoyl)-sn-glycero-3-phosphoethanolamine (DTPE)were purchased from Avanti Polar Lipids. All other chemicals werepurchased from Sigma Aldrich and used without further purification.Dichloromethane (DCM), and toluene were distilled from CaH₂.Triethylamine (TEA) was distilled from CaH₂ and stored over BaO.Tetrahydrofuran (THF) was distilled from sodium-benzophenone ketyl.α-methoxy-polyethylene glycol (mPEG350) was purchased from Sigma Aldrichand purified by azeotropic drying with toluene. Jones'reagent (1.25 M inCrO₃) was prepared by dissolving 17.5 g CrO₃ in 125 mL water plus 16 mLconc. H₂SO₄.

Experimental Methods. Nuclear magnetic resonance spectroscopy (NMR) wasperformed on a Bruker Avance ARX-400 NMR spectrometer using deuteratedchloroform (CDCl₃) as NMR solvent and internal standard unless otherwisenoted.

Example 24 Synthesis of mPEG350-DTPE

mPEG350-CO₂H (2). mPEG350 (1, 5.00 g, 14.3 mmol) was dissolved in 280 mLacetone. Jones' Reagent (15 mL, 1.25 M, 18.75 mmol) was added to a 500mL round bottom flask and the mPEG solution was added to this flaskdropwise over 1 h via addition funnel. The resulting solution wasstirred at 20° C. for 1 h before quenching the excess Jones reagent with10 mL iPrOH. The resulting green precipitate was removed by decantationof the liquid solution. The volatiles were removed under reducedpressure and the residue dissolved in 100 mL H₂O. The aqueous phase wasextracted with DCM (3×120 mL). The organic layers were combined anddried over anhydrous Na₂SO₄, filtered, and evaporated under reducedpressure. The clear oily residue was used without further purification.Yield: 4.52 g (90%); TLC: R_(f)=0.26 (3:17 MeOH:DCM); ¹H NMR (400 MHz,CDCl₃): δ 3.35 (s, 3H), 3.4-3.8 (m, 30H), 4.01 (s 2H), 10.1-12.2 (br1H).

mPEG350-NHS (3). Compound 2 (274 mg, 0.753 mmol), and NHS (217 mg, 1.885mmol) were dissolved in 15 mL DC-M and the resulting solution was cooledin an ice bath. EDC (173 mg, 0.902 mmol) was then added followed by DIEA(393 μL, 2.259 mmol), and the solution was stirred while slowly warmingfrom 4 to 20° C. over 18 h. Volatiles were removed under reducedpressure and the residue was dissolved in 50 mL DCM. The organicfiltrate was washed with 50 mL H₂O twice before combining and extractingthe aqueous phase with DCM (2×20 mL). The combined organic phases weredried over anhydrous Na₂SO₄ before removing the solvent under reducedpressure and purifying the residue by flash chromatography on silica(3:17 MeOH:DCM). Yield: 230 mg (84%); TLC: R_(f)=0.66 (3:17 MeOH:DCM);¹H NMR (400 MHz, CDCl₃): δ 2.65 (s 4H), 3.35 (s 3H), 3.4-3.8 (m 30H),4.01 (s 2H).

mPEG350-DTPE (4). Compound 3 (100 mg, 0.210 mmol) and DTPE (183 mg,0.210 mmol) were dissolved in DMF (5 mL) in a 100 mL round bottom flaskcovered in aluminum foil. DIEA (37 μL) was added and the solution wasstirred at 20° C. for 36 h under N₂. The volatiles were removed underreduced pressure and the residue was purified by chromatography onsilica (3:17 MeOH:DCM). Yield: 82 mg (32%); TLC: R_(f)=0.29 (3:17MeOH:DCM), ¹H NMR (400 MHz, CDCl₃): δ 0.88 (t 6H), 1.25-1.35 (m 44H),1.49 (m 8H), 2.22 (m 8H), 2.53 (br 2H), 2.61 (br 2H), 3.36 (s 3H),3.54-4.32 (m, 40H), 512 (br 1H), 7.64 (br 1H).

Example 25 Synthesis of NTA Derivatives

S-Trityl-3-mercaptopropionic acid (6). 3-Mercaptopropionic acid (5, 6.00g, 56.5 mmol) was dissolved in DCM (50 mL) in a 250 mL round bottomflask. Trityl chloride (17.34 g, 62.2 mmol) in DCM (30 mL) was addeddropwise to this solution over 1 h before stirring for an additional 12h. The white precipitate was filtered and washed with diethyl ether(2×50 mL) and dried under a 50 μm vacuum to give a fine white powder.Yield: 18.25 g (93%); ¹H NMR (400 MHz, CDCl₃): δ 2.25 (t, 2H, J=8 Hz),2.47 (t, 2H, J=8 Hz), 7.2-7.3 (m, 9H), 7.43 (d, 6H).

tert-Butyl N6-((benzyloxy)carbonyl)-1-lysinate (8).N^(ε)-((benzyloxy)carbonyl)-1-lysine (12.03 g, 42.92 mmol) was mixedwith t-butyl acetate (120 mL) in a 250 mL round bottom flask andconcentrated HClO₄ (3.90 mL) added to this mixture, producing a clearsolution. This solution was stirred for 12 h before extracting with 200mL H₂O, 200 mL 5% HCl, then 200 mL, H₂O. The aqueous extracts werecombined and extracted with diethyl ether (3×200 mL) after addition of30% NaOH solution until the aqueous layer was pH 11. The ether extractswere combined and dried over anhydrous MgSO₄. The ether was thenfiltered and concentrated under reduced pressure and dried under a 50 μmvacuum overnight giving a colorless oil. Yield: 9.25 g (63%). ¹H NMR(400 MHz, CDCl₃): δ 1.30 (s 9H), 1.23-1.50 (m 8H), 2.99 (t 2H), 3.11 (t1H), 4.91 (s 2H), 5.61 (br 1H), 7.14-7.16 (m 5H), 13C NMR (101 MHz,CDCl₃): δ 175.18, 156.34, 142.38, 136.60, 128.31, 127.88, 108.60, 80.74,77.46, 77.14, 76.82, 66.29, 54.66, 40.66, 34.36, 31.08, 29.53, 27.90,22.64.

Di-t-butyl2,2′-((6-(((benzyloxy)carbonyl)amino)-1-(t-butoxy)-1-oxohexan-2-yl)azanediyl)diacetate(9). N^(ε)-Benzyloxycarbonyl-L-lysine-t-butyl ester (8, 9.25, 27.5 mmol)was dissolved in DMF (70 mL) prior to the addition of t-butylbromoacetate (12.2 mL, 16.10 g, 82.6 mmol) and DIEA (16.8 mL, 11.9 g,92.1 mmol) by syringe. The solution was stirred under N2 at 70° C. for72 h. The solvent was evaporated under reduced pressure and the residuewas extracted with 200 mL of ethyl acetate and filtered. The ethylacetate extract was purified by flash chromatography on silica (4:1hexane:EtOAc) to give 9 as a slightly yellow oil. Yield: 12.56 g (87%);TLC: R_(f)=0.48 (4:1 hexane:EtOAc); ¹H NMR (CDCl₃): δ 1.25-1.50 (m 6H),1.30 (s 18H), 1.32 (s 9H), 3.04 (m 2H), 3.16 (t 1H), 3.33 (q 4H), 4.93(s 2H), 5.39 (br 1H), 7.15-7.19 (m 5H), 13C NMR (101 MHz, CDCl₃) δ172.07, 170.41, 156.32, 136.69, 128.15, 127.79, 127.64, 80.70, 80.33,77.54, 77.22, 76.90, 66.03, 64.91, 60.04, 53.62, 40.56, 29.93, 29.02,27.97, 27.86, 27.73, 22.80, 20.71, 13.97.

Di-t-butyl2,2′-(6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)azanediyl)diacetate (10).Compound 9 (9.84 g, 17.4 mmol) was dissolved in MeOH (90 mL) in a 500 mLround bottom flask. To this solution was added 40 mg of 10% Pd/C. Theflask was evacuated and purged with H2 three times and then stirred for12 h under 1 atm H2. The heterogeneous solution was then filteredthrough a pad of Celite, with further washing of the Celite cake with 50mL MeOH. The filtrate was concentrated under reduced pressure to give 10as a clear oil. Yield: 7.35 g (98%); TLC: Rf=0 (4:1 hexane:EtOAc); ¹HNMR (CDCl₃): δ 1.29 (s 18H), 1.30 (s 9H) 1.1-1.5 (m 6H), 2.63 (t 2H),3.16 (t 1H), 3.30 (q 4H), 3.96 (br 3H)¹³C NMR (101 MHz, CDCl₃) δ 172.05,170.46, 80.78, 80.42, 77.41, 77.09, 76.77, 64.94, 53.52, 49.54, 40.90,31.09, 30.09, 27.95, 27.87, 22.91.

Di-t-butyl2,2′-(1-(t-butoxy)-1-oxo-6-(3-(tritylthio)propanamido)hexan-2-yl)azanediyl)diacetate(11). Compound 10 (1.46 g, 4.19 mmol) and Compound 6 (1.80 g, 4.19 mmol)were dissolved in DMF (100 mL) in a 250 mL round bottom flask. Thissolution was cooled on an ice bath before addition of EDC (0.962 g, 5.02mmol), HOBt (0.678 g, 5.02 mmol), and 1.86 mL of DIEA (1.35 g, 10.4mmol). The solution was stirred under Ar for 48 h while allowing themixture to gradually warm from 4→20° C. The DMF was evaporated underreduced pressure and the residue was dissolved in EtOAc (80 mL). Thissolution was washed with H₂O (2×100 mL) before combining the aqueousphases and back extracting with EtOAc (100 mL). The EtOAc layers werecombined and dried over anhydrous Na₂SO₄. The solvent was evaporated andthe residue purified by flash chromatography on silica using 1:1hexane:EtOAc as eluent yielding 11 as a colorless oil. Yield: 1.70 g(53%); TLC: Rf=0.54 (1:1 hexane:EtOAc); ¹H NMR (CDCl₃): δ 1.22-1.61 (m6H) 1.39 (s 18H), 1.43 (s 9H), 2.09 (t 2H), 2.46 (t 2H), 3.11 (m 2H),3.25 (t 1H), 3.43 (m 4H), 5.98 (br 1H), 7.14-7.4 (m 15H); MS (ESI+).Expected: 762.02 [M+H]; Found 762.71.

2,2′-(1-Carboxy-5-(3-mercaptopropanamido)pentyl)azanediyl)diacetic acid(12). Compound 11 (0.800 g, 1.05 mmol) was dissolved in DCM (10 mL),followed by addition of Et₃SiH (0.367 g, 3.16 mmol) and2-mercaptoethanol (0.246 g, 3.15 mmol). This solution was cooled on anice bath before addition of TFA (15 mL) dropwise over 10 min. Thesolution was stirred for 1 h at 4° C. before removal of the volatilesunder reduced pressure. Diethyl ether (20 mL) and 4 drops ofconcentrated HCl were added to the residue before decanting the organicphase and repeating the process two more times. Toluene (30 mL) was thenadded to the residue and evaporated under reduced pressure three timesto give compound 12 as a white powder. Yield: 0.327 g (89%); ¹H NMR(D₂O): δ 1.41 (m 2H), 1.49 (m 2H), 1.80-1.90 (m 2H), 2.43 (t 2H), 2.67(t 2H), 2.73 (m 1H), 3.13 (m 2H), 3.93 (s 4H).

Di-tert-butyl-2,2′-((1-(tert-butoxy)-6-(((4-nitrophenoxy)carbonyl)amino)-1-oxohexan-2-yl)azanediyl)diacetate(13). p-nitrophenyl chloroformate (PNP-Cl, 0.552 g, 2.74 mmol) wasdissolved into 20 mL DCM in a 100 mL round bottom flask equipped with astir bar and addition funnel and was cooled to 4° C. in an ice bath.Compound 12 (0.983 g, 2.28 mmol) in 20 mL DCM was added to the additionfunnel. The system was evacuated and flashed with nitrogen gas. Thesolution of 12 was added over a one hour period at 4° C. and stirred foran additional 12 hours warming to room temperature. The solution wasconcentrated in vacuo and purified by flash chromatography using 4:1hexanes: EtOAc as eluent. Yield: 0.609 g (45%); TLC: Rf=0.24 (4:1hexanes: EtOAc); ¹H NMR (CDCl₃): δ 1.38 (s 18H), 1.40 (s 9H), 1.18-1.59(m 6H), 3.18-3.29 (m 3H), 3.40 (q 4H), 6.09 (br 1H), 7.24 (d 2H, J=9Hz), 8.14 (d 2H, J=9 Hz); MS (ESI+). Expected: 596.68 [M+H]; Found596.59 ([M+H], 618.54 [M+Na].

Example 26 Synthesis of NTA-PEG2000-DSPE

Maleimide-PEG2000-DSPE (15). DSPE (0.072 g, 0.096 mmol) andNHS-PEG2000-maleimide (0.200 g, 0.095 mmol) were dissolved in CHCl₃ (15mL) in a 50 mL round bottom, flask with stir bar and DIEA (0.062 g,0.480) mmol was added via syringe. The flask was evacuated and flushedwith nitrogen and stirred for 72 hours at ambient temperature. Thevolatiles were evaporated under reduced pressure and the residuepurified by flash chromatography on silica using a gradient startingwith 85:15 DCM:MeOH and increasing in polarity to 80:20 DCM:MeOH. Yield:0.118 g (43%); TLC: Rf=0.48 (4:1 DCM:MeOH); ¹H NMR (CDCl₃): δ 0.84 (t6H), 1.15-1.40 (m 64H), 2.24 (m 4H), 2.48 (t 2H) 2.99 (m 2H), 3.37-4.10(m 180H), 3.80-3.96 (m 4H), 4.12 (m 2H), 4.34 (d 2H), 5.17 (m 1H), 6.27(br 1H), 6.67 (s 1H), 7.38 (br 1H).

NTA-PEG2000-DSPE (16). Compounds 15 (20.0 mg, 0.007 mmol) and 12 (18.0mg, 0.051 mmol) were dissolved in DMF (4 mL) in a 25 mL round bottomflask with stir bar. TEA (15.0 μL, 0.119 mmol) was added and the flaskwas evacuated and flushed with nitrogen. The solution was stirred at 40°C. for 24 hours monitoring the consumption of starting material by TLC.Volatiles were removed in vacuo at 45° C. the residue was dissolved in 6mL PBS buffer (pH=7.2) plus 4 mL MeOH. This solution was extracted withCHCl₃ (3×15 mL). The organic extracts were combined and dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo to give 16. Yield:21.2 mg (94%); TLC: Rf=0.0 (4:1 DCM:MeOH); ¹H NMR (CDCl₃): δ 0.84 (t6H), 1.15-1.40 (m 64H), 2.25 (s 4H), 2.28-2.32 (m 2H), 2.48-2.53 (m 2H),2.75-2.78 (m 4H), 2.48-3.7 (m 180H), 3.70 (s 4H), 3.88 (br 2H), 4.00 (br2H). 4.16 (br 2H), 4.37-4.40 (br 1H), 5.30 (s 1H), 6.96 (s 1H).

Example 27 Synthesis of NTA-PEG200-DTPE

NHBoc-PEG2000-DTPE (18). NHS-PEG2K—NHBoc (17, 190 mg, 0.095 mmol) andDTPE (82.8 mg, 0.095 mmol) were dissolved in DCM (10 mL) in a 25 mLround bottom flask with stir bar. DIEA (83 μL 0.474 mmol) was added andthe flask was evacuated, flushed with nitrogen and covered with aluminumfoil. The solution was stirred at ambient temperature for 48 hours inthe dark. Evaporated volatiles and purified by flash chromatography onsilica using an eluting system of DCM and MeOH starting with 95:5 then90:10 then 85:15. Fractions containing product were combined and driedin vacuo to give 18. Yield: 0.209 g (88%); TLC: R_(f)=0.72 (80:20DCM:MeOH); ¹H NMR(CDCl₃): δ 0.88 (m 6H), 1.23-1.49 (m 50H), 1.42 (s 9H),2.21 (m 8H), 2.66 (t 4H), 3.26-3.96 (m 180H), 4.11-4.12 (m 2H),4.33-4.36 (m 2H), 5.17 (br 1H).

NH₂-PEG2000-DTPE (19). Compound 18 (209 mg, 0.076 mmol) andtriethylsilane (200 μL, 1.25 mmol) was dissolved in 30% TFA in DCMsolution (20 mL) and stirred for 1.5 hours under ambient temperature andatmosphere. Volatiles were removed in vacuo and the residue wasevaporated with 15 mL DCM twice more. The product was dried in vacuo andused without further purification. Yield: 0.191 g (91%); TLC: R_(f)=0.56(80:20 DCM:MeOH).

NTA-(OtBu)3-PEG2000-DTPE (20). Compound 19 (95.5 mg, 0.034 mmol) andcompound 13 (205 mg, 0.344 mmol) were dissolved in DCM (5 mL) in a 25 mLround bottom flask with stir bar. The flask was evacuated and flushedwith nitrogen. DIEA (60 μL, 0.348 mmol) was added and the solutionstirred for 48 hours at ambient temperature under a nitrogen atmosphere.Volatiles were evaporated under reduced pressure and the product waspurified by flash chromatography on silica using a gradient of DCM:MeOHas eluent starting with 90:10 moving to 85:15 then finally 80:20.Fractions containing product were pooled, concentrated and dried invacuo to give compound 20. Yield: 29.0 mg (27%); TLC: R_(f)=0.65 (80:20DCM:MeOH).

NTA-PEG2000-DTPE (21). Compound 20 (29 mg, 0.009 mmol) andtriethylsilane (100 μL, 0.625 mmol) were dissolved in 30% TFA in DCMsolution (10 mL) in a 25 mL round bottom flask with stir bar. Thesolution was stirred under ambient temperature and atmosphere for 1.5hours. Volatiles were evaporated and the residue was dissolved in 5 mLPBS buffer (pH=7.2) plus 5 mL MeOH. The solution was extracted withCHCl₃ (3×8 mL). The organic extracts were dried over anhydrous Na₂SO₄,filtered, concentrated and dried in vacuo to give compound 21. Yield:18.0 mg (62%; TLC: R_(f)=0.0 (80:20 DCM:MeOH).

The invention claimed is:
 1. A grid for elucidating high-resolutionstructure of target proteins comprising a coating modified with acapture agent and further comprising a deactivating agent, wherein thecoating comprises a graphene oxide sheet modified with Nα,Nα-dicarboxymethyllysine (GO-NTA), wherein the capture agent is Nα,Nα-dicarboxymethyllysine and the deactivating agent ismethoxy-poly(ethylene glycol350)-1,2-distearoyl-sn-3-glycerophosphoethanolamine.
 2. The grid ofclaim 1, wherein the coating is a monolayer.
 3. The grid of claim 1,wherein the coating comprises a plurality of micro-porous or nano-porousgraphene oxide sheets.
 4. The grid of claim 3, further comprising one ormore lipid monolayers wherein the monolayers are deposited onto thegraphenic layer residing on the grid.
 5. The grid of claim 1, whereinthe coating is between about 0.1 nanometers and 100 microns thick. 6.The grid of claim 5, wherein the coating is between about 0.1 nanometersand 100 nanometers thick.
 7. The grid of claim 6, wherein the coating isbetween about 0.1 nanometers and 10 nanometers thick.
 8. The grid ofclaim 7 wherein the coating is between about 0.1 nanometers and 2nanometers thick.
 9. The grid of claim 8, wherein the coating is betweenabout 0.1 nanometers and 1 nanometer thick.
 10. The grid of claim 1,wherein the modification to the coating is done by chemical synthesis.11. The grid of claim 1, wherein the target proteins are tagged withpolyhistidine.
 12. The grid of claim 1, wherein the target proteins areproteins obtained from a cell lysate.
 13. The grid of claim 12, whereinthe cell lysate has been clarified.
 14. The grid of claim 1, whereinsaid grid is used as a substrate for single particle protein structureanalysis by a suitable transmission electron microscope at a resolutionof less than or equal to 20 Angstroms.
 15. The grid of claim 14, whereinthe resolution is between 1 Angstroms and 10 Angstroms.
 16. The grid ofclaim 15, wherein the resolution is between 8 Angstroms and 9 Angstroms.17. A method of preparing target proteins for structure elucidationcomprising contacting a grid of claim 1 with a cell lysate comprisingtarget proteins and subjecting the target proteins to a suitablescanning or transmission electron microscopy for single particlestructure analysis at a resolution of between 1 and 10 Angstroms.